"Frontmatter". In: Plant Genomics and Proteomics

C
LONING
S
YSTEMS
The primary problem of fractionating the genome into manageable bits was
basically solved with the advent of cloning. This methodology, whatever the
vector system used, results in a collection of large numbers of separable frag-
ments. Subsequently, the collection must be screened or additionally char-
acterized to identify the fragments that are of interest. Much of the current
development of new vectors and kits has been done by biotechnology com-
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panies, and the data and protocols are available from their websites. These
developments have made the cloning of both DNA and RNA more routine.
P
LASMID
-B
ASED
V
ECTORS
Most of these cloning vectors are well described and are available in various
forms from the various biotechnology companies. The many plasmid-based
vectors that are available have been engineered for specific tasks, either for
the sequencing or for the expression of the inserted fragment. Included in this
set of specialized vectors are those that also add a short peptide sequence to
the open reading frame to enable protein-protein interactions to be charac-
terized, an example being the yeast two-hybrid systems (more fully described
in Chapter 6) (Bendixen et al., 1994). There are still many uses for plasmid
cloning systems including the generation of small fragments of DNA for
sequencing, the isolation of cDNAs, and especially full-length cDNAs, and
for expressing genes in heterologous systems. The main limitation of plasmid-
based systems is the small size of the insert that can be accommodated. 
One of the more time-consuming processes is the subcloning or shut-
tling of fragments of DNA between different vectors. One of the technolo-
gies that have been developed to facilitate these rearrangements is the
Gateway™ Technology from Invitrogen. Gateway™ Technology is a uni-
versal system for cloning and subcloning DNA sequences, facilitating func-
tional gene analysis and protein expression. Gateway™ Technology enables
the rapid cloning of one or more genes into virtually any protein expression
system. This in vitro technology greatly simplifies the process of gene cloning
and subcloning. As genes are shuttled between expression vectors both the
correct orientation and reading frame are maintained. Gateway™ uses site-
specific recombination, effectively eliminating the requirement to work with
restriction enzymes and ligase after the initial entry clone is constructed.
Once the entry clone has been constructed, the gene of interest can be 
transferred into a variety of Gateway™-adapted expression vectors (desti-
nation vectors). Because the reading frame and orientation of the DNA
fragment are maintained during recombination, the new expression clone
does not need to be sequenced with each new construct. Two reactions, 
BP and LR, constitute the Gateway™ Technology (Figure 2.1). The BP
reaction uses a recombination reaction between an attB DNA segment or
expression clone and an attP donor vector to create an entry clone. The LR
reaction is a recombination between an attL entry clone and an attR desti-
nation vector. The LR reaction is used to move the sequence of interest to
one or more destination vectors in parallel reactions. Constructing a
Gateway™ expression clone is accomplished in just two steps:
1. The gene of interest is cloned into an entry vector via PCR or tradi-
tional cloning methods.
C
L O N I N G
S
Y S T E M S
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2 6
2. T
H E
B
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att 
B
att 
B
gene
att 
P
att 
P
ccd
B
donor
vector
att-flanked
PCR product or
expression clone
att 
L
att 
L
gene
entry
clone
att 
R
att 
R
ccdB
By-product
+
+
BP ClonaseTM
att 
L
att 
L
gene
att 
R
att 
R
ccd
B
expression
clone
entry
clone
att 
B
att 
B
gene
destination
vector
att 
P
att 
P
ccd
B
By-product
+
LR ClonaseTM
FIGURE 2.1.
The r
eactions involved in the Gateway™ T
echnology for moving DNA
sequences between vectors (Copyright 2003 
Invitr
ogen Corporation.
All Rights Reserved. Used W
ith Permission).

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2. The entry clone containing the gene of interest is mixed with the
appropriate destination vector and Gateway™ LR Clonase™ enzyme
mix to generate an expression clone.
L
ARGE
-I
NSERT
V
ECTORS
Three types of vectors that can accommodate large inserts are based on one
of bacteriophage l, yeast artificial chromosomes (YAC) (Kusumi et al., 1993),
and bacterial artificial chromosomes (BAC) (Peterson et al., 2000). Each of
these has its own particular advantages and disadvantages. The l-based
vectors are relatively easy to screen but pose problems in the subsequent
manipulation of each specific recombinant. YACs can accommodate the
largest inserts, but the libraries are difficult to maintain. The ability to store
the BAC clones frozen and to apply automation to the analysis of these
libraries has resulted in a growth in the use of BAC clones. Large-insert
libraries can be used for applications such as the construction of a physical
map and the map-based cloning of genes. For any of the vectors it is impor-
tant to ensure that a sufficiently large and representative library is con-
structed, so that there is a high probability that the particular region of
interest is present in the library. The probability of finding a single copy
sequence in a library is given by: